Transforming growth factor-β1/Smad2/3 signaling pathway plays important roles in osteoblasts differentiation promoted by 0.5 Gy X-ray radiation

Abstract

Objective To investigate the roles of transforming growth factor-β1 (TGF-β1)/Smads signaling pathway in osteoblasts differentiation induced by 0.5 Gy X-ray radiation. Methods The protein levels of osteoblast differentiation markers were detected and matrix mineralization was assessed by Alizarin red staining. Real-time polymerase chain reaction and Western blotting were utilized to evaluate the variations of key factors in TGF-β1/Smads signaling pathways, including TGF-β1, activin receptor-like kinase 5 (ALK5) and Smad2/3. Results The accelerated matrix mineralization was detected after radiation exposure. The protein levels of type I collagen alpha (Col1α), alkaline phosphatase (ALP), osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), Osterix presented dynamic changes after 0.5 Gy X-ray radiation. TGF-β1 mRNA levels increased by 1.7 fold at 4 d after radiation (t=8.414, P=0.000). The mRNA expression levels of ALK5 were increased by 2.2 fold (t=12.660, P=0.000) and 2.5 fold (t=10.380, P=0.000 at 4 d and 7 d after radiation respectively. Enzyme linked immunosorbent assay (ELISA) test show activated TGF-β1 in supernatant was (310.42±36.39) pg/ml (P=0.007) and (420.13±40.61) pg/ml (P=0.000) at 4 d and 7 d after radiation respectively. The increased TGF-β1 activity has a significant difference in radiation group. In addition, p-Smad2 protein level was increased at 7 d after 0.5 Gy X-ray radiation (80.56±7.14, P=0.009). Conclusion TGF-β1/Smad2/3 signaling pathway plays an important regulation mechanism in osteoblast differentiation promoted by low dose X-ray radiation. Key words: X-ray irradiation; Osteoblasts; Cell differentiation; Signal pathway